Directional selection, not the direction of selection, affects telomere length and copy number at ribosomal RNA loci

Many fisheries exert directional selection on traits such as body size and growth rate. Whether directional selection impacts regions of the genome associated with traits related to growth is unknown. To address this issue, we characterised copy number variation in three regions of the genome associated with cell division, (1) telomeric DNA, (2) loci transcribed as ribosomal RNA (rDNA), and (3) mitochondrial DNA (mtDNA), in three selection lines of zebrafish reared at three temperatures (22 °C, 28 °C, and 34 °C). Selection lines differed in (1) the direction of selection (two lines experienced directional selection for large or small body size) and (2) whether they experienced any directional selection itself. Lines that had experienced directional selection were smaller, had lower growth rate, shorter telomeres, and lower rDNA copy number than the line that experiencing no directional selection. Neither telomere length nor rDNA copy number were affected by temperature. In contrast, mtDNA content increased at elevated temperature but did not differ among selection lines. Though directional selection impacts rDNA and telomere length, direction of such selection did not matter, whereas mtDNA acts as a stress marker for temperature. Future work should examine the consequences of these genomic changes in natural fish stocks.

www.nature.com/scientificreports/ in genomic regions that are sensitive to cell division (i.e., growth), such as telomeres, loci that are transcribed as ribosomal RNA, and mitochondrial DNA.
Telomeres are located at the ends of linear (eukaryotic) chromosomes 15 , which in many vertebrates comprise tandem repeats of the motif TTA GGG that are usually about 5-15 kb long 16 .Cell senescence is triggered when a certain proportion of a cell's telomeres become critically short 17,18 and telomere length predicts fitness traits in some animals [19][20][21][22] .Telomeres shorten with cell division 23 unless the telomeres are repaired, for example by telomerase 24,25 .
Loci that are transcribed as ribosomal RNA (hereafter referred to as rDNA) are comprised of tandem arrays of the rRNA cassette (18S, 5.8S, and 28S rRNA loci).Transcription of rDNA is necessary for ribogenesis and protein synthesis.Yet unequal recombination of rDNA can generate a change in rDNA copy number 26 .Indeed, rDNA copy number is sensitive to cell division, as cell division can cause rDNA to become unstable, stimulating molecular aging signals, accelerating cell death, and increasing cancer risk 26 .Interestingly, rDNA exhibits marked intraspecific variation [27][28][29][30][31] , and this may be relevant for adaptation or as a biomarker of health as variation in rDNA copy number apparently regulates gene expression 29,32,33 and affects genome stability 26,[34][35][36] .While the association between rDNA copy number and growth rate is less studied than the association between growth and telomere length, it has been shown that rDNA copy number negatively correlates with body mass in humans and rats 37 .
Mitochondria are membrane-bound organelles that contain their own genome (mitochondrial DNA, mtDNA) and which have essential metabolic and cell-signalling roles, notably by supplying most of a cell's energy requirements 38 .By varying the rate of synthesis and degradation 39 , mitochondrial density in cells (and thus mtDNA content) can vary with age 40 and growth rate 41 .mtDNA depletion can be indicative of cellular malfunction and/or disease 42,43 .It is relevant to quantify mtDNA content in tandem with variation in telomere length and rDNA copy number as (1) mitochondrial content is coupled with rDNA copy number, at least in humans 29 , and (2) mitochondria are a prime source of intracellular reactive species (ROS) (Murphy 2009) that can damage telomeres [44][45][46][47] .
An important feature of these three regions of the genome is that they are sensitive to environment stress.Short telomeres and/or an elevated rate of telomere attrition is a common feature of exposure to stress [44][45][46]48,49 . Likewse, instability in rDNA copy number has been associated with exposure to environmental stress 33,[50][51][52][53] and mtDNA content varies with exposure to pollutants 54,55 .It is therefore relevant to ask whether processes that likely impact copy number, such as directional selection on body size that alters growth, have a concomitant effect on copy number when organisms experience environmental stress.The interaction between environment stress and directional selection induced by fisheries is notoriously difficult to study in nature and thus far only few studies have assessed such interaction 56,57 .
An important environmental stress is thermal stress.For example, extreme weather events can raise sea surface temperature by 2-4 °C, and sometimes > 5 °C58, 59 .Also, temperature regime may affect telomere length, rDNA copy number, and/or mtDNA dynamics, as temperature influences growth rate (i.e.cell division) in many taxa, including teleost fish [60][61][62] .Indeed, temperature effects on telomere length have been reported in teleosts; for example Siberian sturgeon (Acipenser baerii) exhibited a 15% reduction in relative telomere length (RTL) when exposed to 3 °C above ambient 63 , whilst mosquito fish (Gambusia holbrooki) had shorter telomeres at lower temperatures 64 .
Our zebrafish (Danio rerio) selection lines provide an excellent model to determine whether phenotypic differences in body size and growth between lines of fish that experienced directional selection (small and large size) and non-directional selection (random size) associate with differences in telomere length, rDNA copy number, and/or mtDNA content.We also ask whether any differences among lines affect the genomic response to temperature.To address these questions, we exposed young zebrafish (age 50 days) from three selection lines: (1) small-selected fish experiencing directional selection for small body size, (2) large-selected fish experienced directional selection for large body size, and (3) random-selected fish experienced no directional selection, but the lines nonetheless experienced the same reduction in population size during harvesting (see Methods and 2 for more details) to ambient (28 °C), low (22 °C) or elevated (34 °C) temperatures.We hypothesised that (1) directional selection would reduce telomere length and copy number of rDNA and mtDNA compared with random-selection, and (2) that thermal stress would also reduce telomere length and copy number.Furthermore, we predicted that (3) there would be an interaction between thermal stress and directional selection, such that fish exposed to directional selection experience more drastic changes in telomere length and copy number under thermal stress.

Results
Random-selected fish had significantly higher relative telomere length than either small-or large-selected lines (F 2, 247 = 7.09, p < 0.001; Fig. 1a, Table S6).Such differences in telomere length suggests a general effect of directional selection on body size on telomere length rather than any specific effect of the direction (for large or small body size) of selection.Temperature treatment had no significant effect on telomere length, neither was the interaction between selection line and temperature treatment significant in these regions (Table S6).
Similar to relative telomere length, rDNA copy number differed among the three selection lines: randomselected fish had a higher rDNA copy number than fish from either of the size-selected lines, which similarly suggests a general influence of directional selection on rDNA copy number (F 2, 242 = 7.92, p < 0.001; Fig. 1b; Table S6).Also similar to relative telomere length, the temperature treatment did not have a significant effect on rDNA copy number (Table S6), and nor was there a significant interaction between selection line and temperature (Table S6).
In contrast to the pattern of variation in relative telomere length and rDNA copy number, mtDNA content did not significantly differ among the selection lines (Table S6).However, mtDNA content was higher in zebrafish reared at the elevated temperature compared with fish maintained at the low and ambient temperatures (F 2, 262 = 20.2,p < 0.001; Fig. 1c, Table S6).The interaction between the selection line and temperature treatment was not significant (Table S6).
There were significantly positive correlations between relative telomere length and rDNA copy number in all three experimental temperatures (r = 0.366, p < 0.001; Figure S1a, b).A significantly positive correlation between telomere length and rDNA was also observed between all selection lines (r = 0.366, p < 0.001; Figure S1a, b).mtDNA content was negatively correlated with relative telomere length and rDNA copy number in the largeselected line across the three different temperature treatments (mtDNA:rDNA, r = − 0.263, p < 0.05; mtDNA:RTL, r = − 0.256, p < 0.05; Figure S1a).mtDNA was negatively correlated with rDNA copy number at 22 °C across all three selection lines (r = − 0.230, p < 0.05; Figure S1b).

Discussion
Size-selective harvesting impacts diversity at single-copy regions of the genome such as microsatellite loci 7 or SNPs within and among protein-coding regions 6,65 .However, whether size selective harvesting elicits other types of genomic change, such as variation in copy number, and whether any genomic changes affect the response to temperature is not known.Using an experimental zebrafish model, we found that directional selection (for small and large body size) associates with a reduction in telomere length and rDNA copy number, but has no significant effect on mtDNA content, compared to random-selection (i.e., no directional selection).Hence, relative telomere length and rDNA copy number exhibited a correlated response to directional selection, rather than the direction of selection, per se.While mtDNA content was not impacted by directional selection, fish reared at an elevated temperature exhibited an increase in mtDNA content.Counter to our hypothesis we found no evidence of an interaction between directional selection and thermal stress at any of the genomic regions, suggesting an independent action of these processes on copy number variation and telomere length.
Short relative telomere length associating with directional selection on body size is intriguing as, after ten generations of recovery, both directionally selected lines had lower growth rate and reached smaller adult body size than fish which had not experienced directional selection 14 .As telomeres shorten with cell division, unless repaired by telomerase 66 , fast growing, larger individuals (random-selected fish) are expected to have shorter telomeres.Fast growing transgenic coho salmon (Oncorhynchus kisutch) are unable to maintain telomere length 67 , and in brown trout (Salmo trutta), body size (but not compensatory growth) was negatively associated with telomere length 68 and a greater change in telomere length 21 .However, telomere length and expression of telomerase increase with development in zebrafish muscle such that telomeres do not shorten with growth in healthy zebrafish until old age (about 30 months) when telomerase expression declines 69,70 .It could be speculated that www.nature.com/scientificreports/ the fish under directional selection were less capable of telomere maintenance than the line that experienced random selection, however the mechanisms for this would require further studies.Loss of genetic diversity [71][72][73][74] , and potentially inbreeding 75,76 , is a feature of many overharvested fish stocks.Directional selection may elicit a faster loss of genetic diversity than expected under a population reduction alone 77 , as favouring a specific phenotype (e.g., body size) can cause directional shift in allele frequency 78 .Although we do not measure inbreeding or genetic diversity, slower growth associated with directional selection may indicate that these lines experience some inbreeding depression whose effects extend to telomere maintenance 14 .However, the relationship between telomere length and inbreeding is controversial.Studies on wild vertebrate populations have shown that inbreeding/elevated levels of homozygosity is associated with short 79,80 and long telomeres 81 , or have failed to uncover any significant effect of inbreeding on telomere length 82 .Nonetheless, the comparably short telomeres in both size-selected lines indicates that directional selection, at least for body size, can have an unintended, but important outcome on telomere length-an effect that has not been reported in teleost fish.
As it is not possible to quantify changes in telomere length using non-destructive sampling (e.g. from blood 83 ) on such young zebrafish, we do not know whether the outcome of our experiment reflects an inherent difference in telomere length among the lines or whether all fish had similar length telomeres at hatching and the shorter telomeres are a consequence of poor telomere maintenance in the size-selected lines.Nonetheless, that sizeselective harvesting can cause short telomeres and/or poor telomere maintenance is a potential cause for concern for the health of overharvested fish stocks given the widespread reports that short telomeres are a biomarker for stress exposure or reduced health in many animals [19][20][21][22]84,85 including telomerase deficient zebrafish 86 .
Variation in temperature did not impact telomere length in zebrafish in contrast with previous studies demonstrating a negative association between water temperature and telomere length in brown trout (Salmo trutto) 21,68 .In sticklebacks (Gasterosteus aculeatus), variation in temperature did not directly affect telomere length at individual level but had sex specific effects on telomere length in mature fish 87 .This is interesting as old (> 18 months) zebrafish have short telomeres 88 , implying that a longer experimental duration might have revealed additional changes in telomere length in our lines.The lack of interaction between selection line and temperature stress on telomere length is surprising following previous interactions on phenotypic traits including growth 56 .But the lack of interaction is relevant if the size-selected lines experience some inbreeding depression, because a greater impact of inhabiting a poor environment on telomere length was uncovered in less genetically diverse juvenile birds 79 .
As rDNA copy number is sensitive to environment variation 33,50,52,89 it represents a hypothesised 'environmental sensor' that may regulate the molecular response to environmental cues 35,47,90,91 .It is therefore surprising that we found no significant impact of different temperature environments on zebrafish rDNA copy number.Future work could study different species and/or different environmental stressors, such as nutrient stress 92 or exposure to pollutants 52 , to uncover possible drivers of rDNA copy number variation in teleosts.Nonetheless, it is interesting that directional selection on body size affected rDNA copy number as significant differences in rDNA copy number among strains of inbred laboratory mice (Mus musculus 93 ) raise the prospect that rDNA copy number might be impacted by reductions in population size/inbreeding (as discussed for telomere length).Indeed, the significant positive correlation between rDNA and relative telomere length supports the idea that these regions are sensitive to similar stressors 94 .For example, rDNA and telomeres are both sensitive to changes in heterochromatin architecture 95 and oxidative stress 94 .Our data highlight a need to quantify rDNA copy number and telomere length together to determine in what taxa, in what environments, and potentially why, copy number/length of these regions of the genome are co-associated.Moreover, it is important to understand these changes in the context of traits associated with growth as these regions are sensitive to cell division 23,26,66 .The negative association between mtDNA content and rDNA copy number in zebrafish is consistent with the negative association between these regions of the genome in humans 29 .However, the weak relationships between mtDNA content and rDNA copy number likely reflects that mitochondria have a separate genome and mitochondrial density being dynamic and independent of cell division 96 .
The significant effect of temperature on mtDNA content at 34 °C adds to the diversity of biological impacts that occur in aquatic communities exposed to thermal stress.To our knowledge, only one previous study has examined the effect of temperature on mtDNA content in teleosts, in which there was an increase in mtDNA content in eggs at warmer winter temperatures (+ 5 °C) in stickleback (Gasterosteus aculeatus) 97 .Similarly, an increase in mtDNA content was reported in prawns (Palaemon carinicauda) raised in warm water 98 .An increase in mtDNA content corresponds with an increase in mitochondrial content 99 and is consistent with an expected rise in metabolic rate that accompanies an increase in temperature 100,101 .Studying mtDNA content in tandem with telomere length and rDNA copy number is relevant as mitochondrial density may positively associate with the production of reactive oxygen species [102][103][104] that can damage telomeres [44][45][46] and rDNA 105 .For example, an increase in mtDNA density was correlated with production of the free radical superoxide, which in turn influenced telomere length 103 .Indeed, we found negative correlations between mtDNA content and telomere length/ rDNA copy number in the large-selected lines, which (1) reinforces the idea that telomere length (and rDNA copy number) should be quantified in tandem with mtDNA content, and ideally ROS production 103 , and (2) shows how any association between these regions of the genome can depend on genetic background.
Balanced harvesting is hypothesised to mitigate the effects of directional selection 10 , for example by retaining genetic diversity and lessening any effects of inbreeding 11 .Balanced harvesting can increase stock productivity 106 , aid the recovery of a stock's natural size and age structure 107 , and improve the resilience of a stocks to natural disturbance 108 .Here, our random-selected lines experienced harvesting but no directional selection on body size and thus correspond with balanced harvesting.Our zebrafish model of overharvesting supports the idea that balanced harvesting can help maintain growth and body size.We also show how harvesting regime can impact regions of the genome that are associated with organismal health and fitness [19][20][21][22] .We show that directional selection (for body size) has a greater impact on these regions of the genome than an equivalent random reduction in population size.Identifying the mechanisms behind these results requires further work but may be related to a loss of genetic diversity that accompanies directional selection.Quantifying processes that drive variation in telomere length and rDNA copy number is important as maintenance of these loci is thought to be essential to genome integrity 35,109,110 and the rate of molecular aging 89,111 .Intriguingly, our data indicate relative telomere length and rDNA copy number are resilient to changes in temperature.In contrast, mtDNA content was not impacted by directional selection but was increased at elevated temperatures, presumably in response to a change in metabolic requirements.Our data open new avenues for future research of dynamics of telomere length, rDNA copy number, and mtDNA content in wild populations.For example, a next step would be to determine whether natural populations of exploited fish experienced similar genomic impacts and, if so, what are the mechanisms and do these genomic changes alter individual fitness.Crucially, direction of selection (either small-or large-selection on body size) appears less important that the act of directional selection itself, as directional selection reduced rDNA copy number and telomere length regardless of the direction compared to random selection.Our data suggest that selection regimes implicated by fisheries should be reconsidered, utilising alterative harvest strategies such as balanced harvesting to reduce any effects of directional selection on fitness.

Zebrafish model system
Three zebrafish selection lines (two replicates of each line) were created by subjecting wild-caught fish (from the West Bengal region of India 112 ) to the following harvesting regimes: (1) small-selection, where 75% of the largest fish were removed, (2) large-selection in which 75% of the smallest fish were removed, and (3) random-selection (population loss alone), where 75% of the fish were removed at random with regard to body size.After five generations of harvesting, small-selected fish were smaller, had higher juvenile growth rate and higher reproductive investment than large-selected fish 2 .After ten generations of no-harvesting (a 'moratorium'), the random-selected fish had a 12% faster growth rate than both the large-and small-selected lines (which had similar growth rates) 14 .In our model there is a contrast between the large-and small-selected lines that both experienced directional selection on body size in contrast to the random-selected line that experienced the same population reduction (75% harvesting) but no directional selection on body size.All methods were performed in accordance with the relevant guidelines and regulations.All experimental protocols were approved by the Finnish Project Authorisation Board.Licence no.ESAVI/24,875/2018 and all experiments followed the ARRIVE guidelines 113 .

Study design
Fish (n = 243; Table S2) at age 50 days post fertilization were taken from each selection line replicate and exposed to three different temperatures, low (22 °C), ambient (28 °C), and elevated (34 °C), for 250 days.Ambient temperature is the control temperature, as it was the standard rearing temperature in the laboratory for 15 generations as well as representing the natural environment of zebrafish 114 .Elevated and low temperatures were ± 6 °C from ambient, as this presents a physiological stress to zebrafish 115,116 and represents a potential rise in water temperature during extreme weather events 59 .
Fish were housed in 30L glass aquaria with three tanks per temperature treatment.Each aquarium housed eight cylindrical mesh cages with five fish in each.Fish were first acclimated for two weeks (28 °C), after which, the temperature was altered by plus or minus 1 °C per day for six days.At the end of the experiment, fish were euthanized with 2-phenoloxyethanol and stored at -20 until DNA extraction.qPCR to estimate telomere length and copy number DNA was extracted from muscle tissue using a DNeasy blood and tissue kit (Qiagen).DNA quantity was measured using a NanoDrop spectrophotometer (ThermoFisher) and samples were normalised to 5 ng/μl.Relative copy number (analogous to relative telomere length; Cawthon 2002) of each sample was calculated by quantitative PCR (qPCR) using an appropriate locus-specific primer pair and a single copy gene (SCG) (see 117 for relative telomere length 52 , for rDNA, and 55 for mtDNA RCN).
qPCRs were completed on CFX96 thermal cyclers (Bio-Rad) with each qPCR containing 20 ng DNA, 0.3uM of each primer, and 10 μl of iQ SYBR Green supermix (Bio-Rad).Samples were run in triplicate to provide a mean Ct value if the standard deviation (SD) was < 0.2.If a sample qPCR SD was > 0.2, a mean Ct was taken from two qPCRs, or the qPCR was redone in triplicate if the 0.2 SD threshold was not met.Each qPCR plate (that used the same template; Table S3) contained a negative (no DNA) control, the same 'golden standard' DNA (GS), and a serial DNA dilution to calculate qPCR efficiency (1:2 dilution starting from 80 ng/μl).qPCRs were completed on separate plates for the same 26 samples to estimate reproducibility which was high based on Ct values (r > 0.9, p < 0.001 for all loci; Table S4).Full details of the qPCR primers (for each locus and for the SCG), and the thermal cycling conditions are provided in Table S5.Relative copy number (RCN) (or relative telomere length, RTL) were calculated for each sample using: where E(target) and E(control) are the qPCR efficiencies of the target (i.e., telomere, rDNA, and mtDNA) and the single copy gene respectively, and C t GS and C t SAMPLE are the critical cycle thresholds for the golden standard and sample DNAs respectively 117,118 .

Statistical analysis
All statistical analyses were performed in R studio using R v.4.2.2 119 .We used linear mixed models (LMM) models to assess how relative telomere length, rDNA copy number and mtDNA content differed among selection lines and temperature treatments, whereby temperature, selection line and their interaction were included as fixed terms using the following model: where RCN indicates relative telomere length, rDNA copy number, or mtDNA content, and selection-line replicate (n = 2 replicates for each selection line) is a random term.Analyses used the lmer and lmertest functions within lme4 v.1.1-33and lmerTest v.3.1-3packages 120,121 .Pearson's correlation was assessed between pairs of relative telomere length, rDNA copy number, and mtDNA content within each temperature treatment using the cor.test function within GGally v.2.1.2 122.